Frontiers in Immunology (Sep 2021)
Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue
- Chloe M. Doyle,
- Chloe M. Doyle,
- Chloe M. Doyle,
- Chloe M. Doyle,
- Erica E. Vine,
- Erica E. Vine,
- Erica E. Vine,
- Kirstie M. Bertram,
- Kirstie M. Bertram,
- Kirstie M. Bertram,
- Heeva Baharlou,
- Heeva Baharlou,
- Jake W. Rhodes,
- Jake W. Rhodes,
- Jake W. Rhodes,
- Suat Dervish,
- Martijn P. Gosselink,
- Martijn P. Gosselink,
- Martijn P. Gosselink,
- Angelina Di Re,
- Angelina Di Re,
- Angelina Di Re,
- Geoffrey P. Collins,
- Geoffrey P. Collins,
- Geoffrey P. Collins,
- Faizur Reza,
- Faizur Reza,
- Faizur Reza,
- James W. T. Toh,
- James W. T. Toh,
- James W. T. Toh,
- Nimalan Pathma-Nathan,
- Nimalan Pathma-Nathan,
- Nimalan Pathma-Nathan,
- Golo Ahlenstiel,
- Golo Ahlenstiel,
- Golo Ahlenstiel,
- Grahame Ctercteko,
- Grahame Ctercteko,
- Grahame Ctercteko,
- Anthony L. Cunningham,
- Anthony L. Cunningham,
- Andrew N. Harman,
- Andrew N. Harman,
- Andrew N. Harman,
- Scott N. Byrne,
- Scott N. Byrne
Affiliations
- Chloe M. Doyle
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Chloe M. Doyle
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Chloe M. Doyle
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Chloe M. Doyle
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Erica E. Vine
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Erica E. Vine
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Erica E. Vine
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Kirstie M. Bertram
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Kirstie M. Bertram
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Kirstie M. Bertram
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Heeva Baharlou
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Heeva Baharlou
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Jake W. Rhodes
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Jake W. Rhodes
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Jake W. Rhodes
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Suat Dervish
- Westmead Cytometry, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Martijn P. Gosselink
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Martijn P. Gosselink
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Martijn P. Gosselink
- Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia
- Angelina Di Re
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Angelina Di Re
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Angelina Di Re
- Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia
- Geoffrey P. Collins
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Geoffrey P. Collins
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Geoffrey P. Collins
- Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia
- Faizur Reza
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Faizur Reza
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Faizur Reza
- Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia
- James W. T. Toh
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- James W. T. Toh
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- James W. T. Toh
- Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia
- Nimalan Pathma-Nathan
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Nimalan Pathma-Nathan
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Nimalan Pathma-Nathan
- Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia
- Golo Ahlenstiel
- Storr Liver Centre, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Golo Ahlenstiel
- Blacktown Clinical School, Western Sydney University, Blacktown, NSW, Australia
- Golo Ahlenstiel
- Blacktown Hospital, Western Sydney Local Area Health District (WSLHD), Blacktown, NSW, Australia
- Grahame Ctercteko
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Grahame Ctercteko
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Grahame Ctercteko
- Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia
- Anthony L. Cunningham
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Anthony L. Cunningham
- Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Andrew N. Harman
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Andrew N. Harman
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Andrew N. Harman
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- Scott N. Byrne
- Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
- Scott N. Byrne
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
- DOI
- https://doi.org/10.3389/fimmu.2021.727952
- Journal volume & issue
-
Vol. 12
Abstract
The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.
Keywords
